Characterization and Crystallization of Soluble Human Fcγ Receptor II (CD32) Isoforms Produced in Insect Cells

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Abstract
FcγRII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the FcγRII:IgG complex, we produced both FcγRII isoforms (FcγRIIa and FcγRIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated FcγRII to IgG. The dissociation constant of the sFcγRIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 × 10-7 M). Complementary sFcγRIIa antagonizes immunocomplex binding to B cells. Here sFcγRIIa showed a comparable dissociation constant (KD = 1.7 × 10-7 M) which was almost 10-fold lower than the constant for FcγRIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two FcγRII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-FcγRII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the FcγRII−ligand interaction, we crystallized FcγRIIb. Trigonal crystals diffracted to 3 Å and the structure solution is in progress.